Swiss albino male mice (6 weeks of age) of average body weight were purchased from Animal Division of International Center for Diarrhoeal Disease Research, Bangladesh (ICDDR, B). The mice were randomly selected and kept in plastic cages with wood-cobe bedding (5 mice/cage). After five days of acclimation, mice were divided into four groups namely control, hyacinth (Hy), arsenic (As) and arsenic-hyacinth (As+Hy). Control mice were maintained with available supply of distilled water and normal mice feed. Hy was provided with HRP containing feed (8% wt/ wt) and distilled water. Arsenic group was given normal feed and sodium arsenite (NaAsO₂) containing water (0.2 g/L). The fourth group, As+Hy group was provided with HRP containing feed and arsenic containing water. These different groups of mice were maintained for 8 weeks. All these procedures and experiments using mice were undertaken following the ethical issues set by the Faculty of Biological Sciences, University of Dhaka, Bangladesh.

Water hyacinths were collected from local lakes around Dhaka, Bangladesh and then the root parts were taken. The roots were washed several times with clean water, and finally degerminated through washing with chlorine containing water. The roots were then cut into smaller pieces and sun dried for a week. HRP were obtained by grinding the dried roots. This powder (8% wt/wt) was mixed with mice feed purchased from ICDDR, B.

Each mouse of a group was weighed every 2 weeks after starting the respective diet and recorded accordingly. A scale in cm was used to measure the tail length.

Surgical blade (size 11) was pinched sharply between ear and eye of mice. Blood came out as drops and collected in test tubes. Blood films were made by placing a drop of blood on one end of a slide, and using a spreader slide blood drop was dispersed over the slide's length. The slide was left to air dry, and fixed immersing it in methanol for 3 to 5 min. After fixation, the slide was stained with Leishman's stain (a mixture based on methylene blue and eosin) to distinguish the cells from each other. Blood cell morphology was then observed under inverted microscope (Kruss Optronic, D22297, Germany). Serum was prepared from collected blood, and LDH, ALP and SGPT were measured using comercially available assay kit (DiaSys Diagnostic Systems, Turkey; Biosystems S.A., Spain; and Human Diagnostic, Germany).

Data were statistically analyzed using Student's t-test with GraphPad Prism 5.

Some previous studies reported that arsenic reduced normal growth and caused loss of body weight and tail wounding in experimental mice (25, 26). Here, we investigated whether HRP could rescue the loss of body weight and other physical damage in mice caused by arsenic. At the beginning of the experiment, the average mice weights of control, hyacinth (Hy group), arsenic (As group) and arsenic-hyacinth (As+Hy group) were 30.12± 0.58, 30.3±0.57, 30.82±1.17 and 30.65±0.73g, respectively.

Weight of the mice in each groups were taken up to 8 weeks at every 2 weeks interval. After 8 weeks, the average body weight of control, Hy, As and As+Hy group were found 43.4±1.03, 42±1.84, 24.09±1.19 and 30.7± 1.12 g, respectively (Figures 1 and 2). The average body weight of the control mice was increased from 30.12 to 43.4 g over 8 weeks. This normal increase of mice body weight was retarded after drinking arsenic containing water. A gradual weight loss in As group of mice was observed during those 8 weeks indicating arsenic-mediated growth retardation. Interestingly, HRP partially rescued growth retardation in arsenic exposed mice when supplemented in feed. Body weight of As+Hy mice was significantly higher at each 2 weeks interval (**p < 0.05) (Figure 1). The growth pattern of control and Hy groups were found to be similar indicating that HRP alone had no visible effects on the growth of the mice.

Mice of each group were also investigated frequently to observe other physical changes caused by arsenic. Arsenic exposed mice were found relatively restless compared with control mice. After four weeks, As-group mice developed wound at their tail tips. The tails became gradually shorter due to cell death at the tail tip (Figure 3). As-Hy group of mice also showed tail reduction, however, this reduction was significantly less than that of As-group mice. This indicated that HRP might have some effects to neutralize arsenic toxicity that caused tail wounding.

The blood cells were then observed under microscope to see the effect of arsenic on cellular morphology. The blood cells of control group were found spherical in shape (Figure 4). However, this normal spherical shape of the cells was distorted in blood collected from arsenic exposed mice. Surprisingly, this distortion of cellular morphology was almost blocked in As-Hy mice. This result suggested that HRP could reduce arsenic-mediated stress causing distortion of blood cell morphology.

Some previous reports showed that arsenic could affect heart and liver tissues and increase the level of LDH, ALP and SGPT in serum (7, 9–11, 27, 28). We next investigated whether HRP could reduce the elevation of these serum enzymes in mice caused by arsenic. In arsenic-exposed mice, serum LDH level was found almost double (1268.15±18.98 U/L) when compared with control mice (686.5± 17.04 U/L) (Figure 5). This result indicated possibilities of heart and other tissue damage in arsenic-exposed mice that might elevate the level of LDH in the serum. However, this serum LDH elevation was partially blocked (1189.88±22.84 U/L) in As-Hy group mice. The reduction of the enzyme level was found significant (**p < 0.05) compared to arsenic-exposed mice (Figure 5).

As-mice also showed a significant increase (***p < 0.05) in serum ALP (458.5±11.6 U/L) and SGPT (152.02±10.43 U/L) levels compared to control (305.6±11.0 and 92.14±5.11 U/L, respectively). Increased levels of ALP and SGPT refer that arsenic might cause liver damage resulting in release of these enzymes into the serum. Again, in case of As-Hy mice, HRP was found to partially reduce the elevation of serum ALP (409.25±19.15 U/L) and SGPT (126.75±10.14 U/L) values.